ES2363228T3 - GLICOSILATED PROTEIN TEST PROCEDURE. - Google Patents

Abstract

The present invention provides a convenient, efficient method for assaying glycated protein, fructosyl peptide, or fructosyl amino acid which can be performed with reduced effect of fructosyl lysine compounds. The invention also provides a reagent for the assay. The invention is directed to a method for reducing the effect of a fructosyl lysine compound in an assay of fructosyl peptide or fructosyl amino acid contained in a sample, characterized by including causing an enzyme for assaying fructosyl peptide or fructosyl amino acid to act specifically on fructosyl peptide or fructosyl amino acid at a pH of 4.0 to 7.0 and measuring the product at a pH of 4.0 to 7.0; and a method for assaying glycated protein through the above method.

Description

Technical field

The present invention relates to a method for testing glycosylated protein, glycosylated peptide or glycosylated amino acid contained in a sample.

Prior art

The glycosylated protein is a non-enzymatically glycosylated protein that is an Amadori compound that is formed from Amadori transposition after the formation of a Schiff base between the aldehyde group of a sugar and the amino group of a protein. Glycosylated proteins are ubiquitously present in the living body. The level of glycosylated protein concentration in blood is greatly influenced by the concentration of monosaccharide (eg glucose) dissolved in serum. Exemplary glycosylated proteins include proteins whose α-amino group at the amino terminus is glycosylated (for example, glycosylated hemoglobin) and proteins whose ε-amino groups contained in internal lysine residues are glycosylated (for example, glycosylated albumin). The concentration of serum glycosylated albumin or the ratio of glycosylated hemoglobin with respect to non-glycosylated hemoglobin present in erythrocytes reflects the average sugar level over a certain period and, therefore, is used as an index for clinical diagnosis such as diagnosis of diabetes, control of the pathological condition or judgment of the therapeutic effect.

Among the glycosylated protein assays there is a procedure called "enzymatic procedure" that can be performed easily and is adaptable to an automatic analyzer commonly used in clinical laboratories (for example, Patent Document 1). The enzymatic process consists of a pretreatment stage necessary for the reaction of a protease with glycosylated protein contained in a sample in order to release the glycosylated peptide or glycosylated amino acid, which serves as a substrate in the next stage, and a test stage necessary for the Reaction of the free substrate with a glycosylated peptide specific enzyme or a glycosylated amino acid specific enzyme (for example, oxidase) in order to produce a detectable substance (for example, hydrogen peroxide) and measure the substance. A glucose oxidase can be reacted with a released saccharide to produce hydrogen peroxide that can be used to measure glycosylated protein with high sensitivity (Patent Document 6).

However, there has been a problem with the enzymatic procedures reported to date since a protease used in the pretreatment stage and a specific enzyme used in the test stage do not have sufficient capacity to recognize differences between glycosylated protein, glycosylated peptide and amino acid glycosylated (hereinafter collectively referred to as "glycosylated protein or the like") and, therefore, if the target glycosylated protein coexists with another glycosylated protein that has not been chosen as the target for the assay, both proteins react with the specific enzyme and thus the specificity of said procedure is weakened.

Examples of glycosylated peptide specific enzyme or glycosylated amino acid specific enzyme that is used in the pretreatment stage include an oxidase that is produced from a bacterium belonging to the genus Corynebacterium (see, for example, Patent Document 2) and a oxidase that is produced from a bacterium belonging to the genus Aspergillus (see, for example, Patent Document 3). These enzymes react effectively with glycosylated amino acid, but tend to remain unreacted against the glycosylated peptide. Recently an enzyme has been reported that is obtained by modifying a fructosylamino acid oxidase (Patent Document 4) and a fructosyl peptide oxidase derived from a filamentous fungus (Patent Document 5), in addition to two fructosyl peptide oxidases of fungi (Non-patent document 8). It is reported that these oxidases react specifically with a glycosylated peptide, especially fructosylvalylhistidine, at pH 8.0. An assay for fructosyl valine at pH ≤6 using a fructosyl amino acid oxidase is described in Patent Document 7.

In the measurement of glycosylated hemoglobin through the use of such a fructosyl amino acid oxidase, the specificity of the oxidase is derived from the difference in the reactivity of the oxidase with fructosyl valine (the valine at the amino end of the hemoglobin subunit β is glycosylated) or fructosyl-valylhistidine (an amino acid residue is additionally added to fructosyl-valine) and with ε-fructosyl-lysine (the lysine residue (s) in a protein is glycosylated (s) ). A hemoglobin molecule has 44 lysine residues. Therefore, even when the reactivity of an oxidase with ε-fructosyl-lysine is low, the total effect of ε-fructosyl-lysine residues should not be ignored. In addition, a compound that contains fructosyl-lysine and is derived from hemoglobin or other plasma proteins (for example, glycosylated albumin) or is derived from a food or drug (hereinafter referred to as "fructosyl-lysine compound") has to be carry a risk that could lead to a positive error. Such a risk is difficult to avoid while using a conventional test procedure.

[Patent document 1] JP-A-1999-127895 [Patent document 2] Japanese patent publication (kokoku) No. H5-33997 (1993)

[Patent document 3] JP-A-1991-155780 [Patent document 4] JP-A-2001-95598 [Patent document 5] JP-A-2003-235585 [Patent document 6] JP-A-2000 -333696 [Patent document 7] JP-A-2001-204494 [Non-patent document 8] Hirokawa, K., Gomi, K. and Kajiyama, N. (2003) Biochem. Biophys Res. Commun. 311, 104-111

Disclosure of the invention

Problem (s) to be solved by the invention

Accordingly, the present invention provides a convenient efficient method for testing glycosylated protein, fructosyl-peptide or fructosyl-amino acid, which can be applied to a variety of automatic analyzers while reducing the effect of fructosyl-lysine compounds, and a reagent. employed in the procedure.

Means to solve the problem

In view of the foregoing, the present inventors have conducted extensive studies and have found that, in the glycosylated, fructosyl-peptide or fructosyl-amino acid protein assay, the effect of the fructosyl-lysine compounds present in the reaction system can be reduced causing an enzyme to test fructosyl-peptide or fructosyl-amino acid (hereafter referred to as "test enzyme") act on fructosyl-peptide or fructosyl-amino acid at a pH of 4.0 to 7.0. The present invention has been made based on this finding.

The present invention provides a method for reducing the effect of a fructosyl-lysine compound in the fructosyl-peptide or fructosyl-amino acid assay, characterized in that it comprises having a fructosyl-peptide oxidase act on fructosyl-peptide or fructosyl-amino acid at a pH. from 4.0 to 7.0 and measure the resulting product at a pH of 4.0 to 7.0.

The present invention also provides a method for reducing the effect of a fructosillisin compound in the glycosylated protein assay of a sample, characterized in that it comprises treating the sample containing the glycosylated protein with a protease so as to release fructosyl peptide or fructosyl amino acid. free, make a fructosyl peptide oxidase act on the release of fructosyl peptide or fructosyl amino acid at a pH of 4.0 to 7.0 and measure the resulting product at a pH of 4.0 to 7.0.

A reagent for testing glycosylated protein with reduced effect of a fructosillisin compound containing at least (A) a protease, (B) a fructosyl peptide oxidase acting on fructosyl peptide or fructosyl amino acid at a pH of 4.0 to 7.0 to produce hydrogen peroxide, and (C) a reagent for measuring hydrogen peroxide.

The present invention also provides a method for reducing the effect of a fructosillisin compound in the fructosyl-peptide or fructosyl-amino acid assay, characterized in that it comprises causing at least the following (A) to (C) to act on fructosyl-peptide or Fructosyl amino acid at a pH of 4.0 to 7.0: (A) a fructosylpeptide oxidase, (B) a reagent for measuring hydrogen peroxide, and (C) an oxidant and glucosone decomposition enzyme.

The present invention also provides a method for reducing the effect of a fructosillisin compound in the glycosylated protein assay contained in a sample, characterized in that it comprises treating a sample containing the glycosylated protein with a protease to thereby release fructosyl-peptide or fructosyl- amino acid and make at least the following (A) to (C) act on the release of fructosyl-peptide or fructosyl-amino acid at a pH of 4.0 to 7.0: (A) a fructosyl-peptide oxidase, ( B) a reagent for measuring hydrogen peroxide, and (C) an oxidant and glucosone decomposition enzyme.

Effects of the invention

The present invention provides an accurate test for glycosylated protein, fructosyl-peptide or fructosyl-amino acid, while reducing the effect of fructosyl-lysine compounds. The process according to the present invention can be performed by a simple operation and, therefore, can be applied to various analysis procedures. Therefore, the process of the present invention is very useful in clinical trials.

Brief description of the drawing

[Fig. 1] Fig. 1 shows the correlation between the hemoglobin A1c (%) values obtained from the process of the present invention (Example 5) and the hemoglobin A1c (%) values obtained from a reference procedure, in addition to the correlation between the hemoglobin A1c (%) values obtained from the procedure of Comparative Example 5 and the hemoglobin A1c (%) values obtained from the reference procedure.

Best way to carry out the invention

There is no particular limitation on the glycosylated protein to be tested while the glycosylated protein is formed by non-enzymatic binding of a protein with an aldose such as glucose. Examples of the glycosylated protein include glycosylated hemoglobin and glycosylated albumin. Of these, glycosylated hemoglobin, especially hemoglobin A1c (HbA1c), is preferred.

Examples of a biological sample containing glycosylated protein include whole blood, hemocytes, serum, plasma, cerebrospinal fluid, sweat, urine, tear fluid, saliva, skin, mucosa and hair. Glycosylated protein is also contained in a wide range of foods such as juice and condiments. Of these, whole blood, hemocytes, serum and plasma are preferred. The sample can be measured as such; that is, without undergoing any treatment. Alternatively, before measurement, the sample can be filtered or dialyzed. In addition, the glycosylated protein to be measured can be properly subjected to concentration, extraction or dilution with water or a buffer, which is described later in this document.

In the process of the present invention, first, the glycosylated protein is treated with a peptidase to release fructosyl-peptide or fructosyl-amino acid. No particular limitation is imposed on the protease while the protease has proteolysis or peptidolysis activity. Preferably a protease is used that can efficiently release fructosyl-peptide or fructosyl-amino acid (preferably fructosyl-valylpeptide or fructosyl-valine) in a short period of time. No particular limitation is imposed on the origin of the protease, and the protease can be derived from a microorganism, an animal, a plant or the like. Specific examples of the protease include commercially available reagents for study purposes such as proteinase K, trypsin, bromelain, carboxypeptidase, papain, pepsin and aminopeptidase; and commercially available reagents for industrial use such as neutral proteinase, Toyozyme NEP (these two are products of Toyobo, Ltd.), acid protease, alkaline protease, Molsin, AO protease, peptidase (these five are products of Kikkoman Corporation), Sumizyme CP , Sumizyme TP, Sumizyme LP50D (these three are products of Shin Nihon Chamical Co., Ltd.), Thermoase PC10F, Protin PC, Protin PC10F, Protin PS10, Protin NY10, Protin NL10, Protin NC25 (these seven are products of Daiwa Kasei KK), Actinase AS (product of Kaken Pharmaceutical Co., Ltd.), Pronase E (product of Roche), Umamizyme, protease S "Amano" G, protease A "Amano" G and protease P "Amano" 3G (these four are products of Amano Enzyme Inc.). The protease effect can be determined as follows: the protease is reacted with fructosyl peptide

or the like, the glycosylated protein or fructosyl peptide before the reaction and the resulting product after the reaction are subjected to capillary electrophoresis, and the results are analyzed and compared between before and after the reaction with the protease. The protease can be used individually or in combination of two or more species. The protease is preferably derived from a microorganism belonging to the genus Bacillus, Aspergillus or Streptomyces; or is produced from a gene of such a microorganism; or belongs to metalloproteinase, neutral protease, acid protease or basic protease. Examples of enzymes derived from the genus Bacillus include Protin PC10F, Protin NC25 (these two are products of Daiwa Kasei K.K.) and Toyozyme NEP (product of Toyobo, Ltd.). Examples of enzymes derived from the Aspergillus genus include Molsin (product of Kikkoman Corporation). Examples of enzymes derived from the genus Streptomyces include Actinase AS, Actinase AF, Actinase E (these three are products of Kaken Pharmaceutical Co., Ltd.) and protease type XIV (product of Sigma). These enzymes can be used individually or in combination (for example, combination of proteinase K and Toyozyme NEP, etc.).

No particular limitation is imposed on the concentration of the enzyme during actual use while the protease can efficiently release a free fructosyl peptide of interest or a free fructosyl amino acid of interest. An appropriate concentration of the protease can be determined experimentally considering the specific activity

or a similar protease factor. For example, the protease derived from the Aspergillus genus (for example, Molsin, product of Kikkoman Corporation) is added in a concentration of 0.0001 to 50 mg / ml, preferably 0.001 to 20 mg / ml. The pH adjustment for the protease treatment is not necessarily performed, but the pH can be adjusted to a value that is optimal for the enzyme reaction, within a range of 3.0 to 11.0. The pH adjustment is carried out by the use of a suitable pH regulating agent such as a buffer, which is described later in this document. The treatment temperature is preferably 10 to 40 ° C.

No particular limitation is imposed on the buffer. Examples of the buffer include phosphate, phthalate, citrate, Tris, maleate, succinate, oxalate, tartrate, acetate and Good buffers (MES, PIPES, ADA, etc.). No particular limitation is imposed on the buffer concentration. The buffer concentration may be 0.00001 to 2 mol / l, preferably 0.001 to 1 mol / l.

Examples of a free fructosyl-peptide or a free fructosyl-amino acid that are obtained by treating a sample with a protease include fructosyl-valylhistidine and fructosyl-valine. A biological sample or a food product before being treated with a protease already contains fructosyl-peptide or fructosyl-amino acid, which is formed by binding glucose to the free peptide or amino acid released by the digestion of glycosylated protein and by the transposition of Amadori of the product. These fructosyl peptide and fructosyl amino acid are also included in the free fructosyl peptide or fructosyl amino acid that is reacted with a test enzyme.

The free fructosyl peptide or free fructosyl amino acid can be tested by reacting a test enzyme with fructosyl peptide or fructosyl amino acid and then measuring the substance obtained from the reaction.

No particular limitation is imposed on the test enzyme that is reacted with fructosyl peptide or fructosylamino acid while the test enzyme is a fructosyl peptide oxidase (JP-A-2001-95598 and JP-A-2003-235585 ). The enzyme can be derived from a microorganism, an animal, a plant, etc., or it can be genetically engineered. In addition, the enzyme may or may not be chemically modified. The enzyme may be in the form of a solution or dry form, and may be supported on or attached to an insoluble vehicle. The enzyme can be used individually or in combination of two or more species.

The amount of enzyme used varies depending on the type of enzyme. The enzyme is preferably used in an amount of 0.001 to 1,000 U / ml, particularly preferably 0.1 to 500 U / ml. The enzyme concentration can be determined experimentally appropriately considering the specific activity of the enzyme. The pH conditions under which the enzyme is reacted are determined using a buffer considering the optimal pH of the enzyme, and adjusted to pH 4.0 to 7.0, preferably pH 4.0 to 6.7, at which the effect of the present invention can be guaranteed. The reaction temperature can be appropriately selected from a temperature range that is generally used in enzymatic reactions; for example, 10 to 40 ° C. As before, no particular limitation is imposed on the buffer. Examples of the buffer include phosphate, phthalate, citrate, Tris, maleate, succinate, oxalate, tartrate, acetate and Good buffers. Of these, phosphate, citrate and ADA (N- (2-acetamide) iminodiacetic acid) buffers are preferred because they guarantee excellent stability during the storage of enzymes for the measurement of fructosyl-peptide or fructosyl-amino acid. No particular limitation is imposed on the buffer concentration, and the concentration is preferably 0.00001 to 2 mol / l, more preferably 0.001 to 1 mol / l.

Depending on the needs, the above enzyme for measurement can be used in combination with another enzyme, a coenzyme, an oxidizing producer reagent, etc. Examples of the enzyme include peroxidase, diaphorase and amino acid metabolizing enzymes that do not react with a substrate, fructosyl valine. Alternatively, an enzyme such as ascorbic acid oxidase or bilirubin oxidase can be used to treat other components present in the blood. Examples of coenzyme include nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide reduced (NADH), nicotinamide adenine dinucleotide phosphate (NADP), nicotinamide adenine adinucleotide phosphate reduced (NADPH), thio-NAD and thio-NAD .

No particular limitation is imposed on the oxidizing producer reagent while the oxidizing producer reagent is reacted with hydrogen peroxide in order to reveal the color. Examples include a combination of 4-aminoantipyrine and a phenol compound, a naphthol compound or an aniline compound, and a combination of 3-methyl-2-benzothiazolinone hydrazone and an aniline compound. Examples of the phenol compound that can be combined with 4-aminoantipyrine include phenol, p-chlorophenol, 2,4-dichlorophenol, 2,4-dibromophenol and 2,4,6-trichlorophenol. Examples of the aniline compound that can be combined with 4-aminoantipyrine include N, N-dimethylaniline, N, N-diethylaniline, N, N-dimethyl-m-toluidine, N, N-diethyl-m-toluidine, N-ethyl-N-sulfopropyl -m-toluidine, N-ethylN- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS), N-ethyl-N- (3-methylphenyl) -N'-acetylethylenediamine, 3-methyl-N-ethyl -N (hydroxyethyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline (ALOS), N-ethyl-N- (3-sulfopropyl) aniline (ALPS), N, N-dimethylm-anisidine and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-anisidine (ADOS), etc. Other examples include N (carboxymethylaminocarbonyl) -4,4'-bis (dimethylamino) -diphenylamine · sodium salt (DA-64), 10- (carboxymethylaminocarbonyl) 3,7-bis (dimethylamino) -phenethiazine · sodium salt (DA -67), 10-N-methylcarbamoyl-3,7-dimethylamino-10H-phenothiazine (MCDP), N, N, N ', N', N '', N '' - hexa-3-sulfopropyl-4,4 ', 4' '- triaminotriphenylmethane (TPM-PS), diaminobenzidine, hydroxyphenylpropionic acid, tetramethylbenzidine and orthophenylenediamine, etc.

Examples of a substance produced by reacting the test enzyme with fructosyl-peptide or fructosylamino acid include peptide, hydrogen peroxide and glucosone, etc. The free fructosyl peptide or fructosylamino acid test can be performed by measuring such a substance produced. Of the compounds listed, hydrogen peroxide is preferred since the measurement can be performed in a simple manner in a short period of time. Hydrogen peroxide can be determined by, for example, a procedure using an oxygen electrode and an enzymatic process using both a peroxidase and a diaphorase and the above coloring agent, the latter being preferred.

Normally, the measurement of hydrogen peroxide is carried out directly after the reaction stage of the test enzyme with fructosyl-peptide or fructosyl-amino acid to produce hydrogen peroxide. The pH of the solution in which the hydrogen peroxide is to be measured is adjusted with a buffer at a pH of 4.0 to 7.0, preferably 4.0 to 6.7. The degree of color developed (change in absorbance) is measured by means of a spectrophotometer and compared with one of a fructosyl-peptide or standard fructosyl-amino acid or the like having a known concentration. Therefore, the glycosylated, fructosyl-peptide or fructosylamino acid protein contained in a sample can be determined.

The process for testing the glycosylated protein according to the present invention includes a step of releasing free fructosyl-peptide or fructosyl-amino acid and a step of measuring the release of free fructosyl-peptide or fructosylamino acid. In the present invention, these steps can be performed separately, or they can be performed in a single procedure; that is, performed seriously and directly one after the other.

In the process for testing the glycosylated protein according to the present invention, the pH of the reaction mixture or the solution to be measured is adjusted to 4.0 to 7.0, preferably 4.0 to 6.7. This adjustment reduces the effect of the fructosyl-peptide or fructosyl-amino acid test enzyme on fructosyl-lysine compounds. Therefore, adjusting the pH to be within the previous range is essential in the step of measuring fructosyl-peptide or free fructosyl-amino acid.

The reagent for testing glycosylated protein that provides reduced effect of fructosyl-lysine compounds includes at least (A) a protease, (B) a fructosyl-peptide-oxidase that produces hydrogen peroxide acting on fructosyl-peptide or fructosyl-amino acid at a pH 4.0 to 7.0, and (C) a reagent for determining hydrogen peroxide. In addition, the sensitivity of the assay can be enhanced by reacting the oxidase described above in (B) with fructosyl-peptide or fructosyl-amino acid to produce glucosone and reacting an oxidative and decomposing enzyme of glucosone with glucosone in order to produce hydrogen peroxide ( JP-A-2000-333696). The glucosone oxidant and decomposition enzyme is preferably at least one oxidase selected from the group consisting of glucose oxidase, β-galactosidase and pyranose oxidase. Enzymes and reagents are as described above. In addition, when glycosylated hemoglobin, especially hemoglobin A1c, is tested, a pretreatment agent that releases hemoglobin from erythrocytes can be used to provide hemoglobin for the assay or a similar agent. In addition, an enzyme can be incorporated into the reagent to treat impurities contained in the blood; a reaction regulating agent; a stabilizer for the reagent; a salt such as sodium chloride, potassium chloride or potassium ferrocyanide; a tetrazolium salt to eliminate the effect of a reducing substance; an antibiotic that serves as a preservative; sodium azide; etc.

Examples of the pretreatment agent include anionic surfactants and non-ionic surfactants (eg, Triton X-100) selected from polyoxyethylene derivatives. The amount of surfactant used is from 0.0001 to 10%, preferably from 0.001 to 1%, with respect to the sample, which according to the needs has been subjected to filtration, dialysis or similar treatment.

The reagent for testing glycosylated protein can be provided not only as a solution, but also in a dry form or as a gel. The reagent can be packaged in a glass vial, a plastic container, etc., or it can be applied to or impregnated in an insoluble vehicle. Examples of the insoluble vehicle include particulate / spherical vehicles made of, for example, latex, glass and colloid; flat plate vehicles made of, for example, semiconductor or glass; and film-like or filament-like vehicles such as those made of paper or nitrocellulose.

Examples

The present invention will now be described in more detail by examples that should not be construed as limiting the invention itself.

[Example 1] Fructosyl-amino acid measurement

(one)

 Preparation of sample

Fructosyl-valine (fV), fructosyl-valylhistidine (fVH) (these two are products of BioQuest) and ε-fructosyl-lysine (fK) (product of Kikkoman Corporation) were dissolved individually in 20 mmol / l phosphate buffer (pH 7.0) to prepare samples (0.3 mmol / l each).

(2)

 Sample Measurement

The prepared samples were measured by means of a HITACHI 7150 automatic analyzer according to the following procedure.

<First reagent>

0.1 mol / l phosphate buffer

Example 1: pH 5.5, pH 6.0, pH 6.5, pH 7.0 Comparative Example 1: pH 7.5, pH 8.0

<Second reagent>

5 U / ml fructosyl peptide oxidase (FPOX-CE, product of Kikkoman Corporation) 20 U / ml peroxidase (III) (product of Toyobo, Ltd.) 1 mmol / l of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine

(TOOS) 1 mmol / l of 4-aminoantipyrine 0.1 moles of phosphate buffer

Example 1: pH 5.5, pH 6.0, pH 6.5, pH 7.0 Comparative Example 1: pH 7.5, pH 8.0 5 The first reagent and the second reagent used the same pH for the assay.

Each 240 μl of the first reagent was added to each 20 μl of the samples, and the resulting mixture was incubated at 37 ° C for 5 minutes. Subsequently the absorbance of the mixture (absorbance I) was measured. Then, the second reagent (80 µl) was added further thereto, and the resulting mixture was incubated at 37 ° C for 5 minutes. Subsequently the absorbance of the mixture was measured (absorbance II). The absorbance was measured at the dominant wavelength of 546 nm and the non-dominant wavelength of 800 nm. The above procedure was repeated using physiological saline instead of the samples, and the resulting mixture was used as a control (reagent containing no fV, fVH or fK (hereinafter referred to as reagent blank)). For each sample, the difference between the

Absorbance I and absorbance II (measurement value) were calculated by the following equation A.

Equation A: Difference in absorbance (measurement value) = Absorbance II - (Absorbance I x (20 + 240) / (20 + 240 + 80))

20 Based on the measurement values obtained, the fK / fV values (ratio of the measurement values of ε-fructosyl-lysine samples with respect to the measurement values of fructosyl-valine samples) and the values of fK / fVH (ratio of the measurement values of ε-fructosyl-lysine samples to the measurement values of fructosyl-valylhistidine samples). The fK / fV and fK / fVH values measured at various pH conditions were compared with those measured at a pH of 8.0 (100%). The results are shown in Table 1.

25 [Table 1]

pH of the reaction mixture

Example 1 Comparative Example 1

5.5

6.0 6.5 7.0 7.5 8.0

Ratio of measurement values: fK / fV (%)

0.8 2.1 5.7 12.8 23.8 32.3

Percentages with respect to fK / fV (pH 8.0) (%)

2.5 6.5 17.6 39.6 73.7 100.0

Ratio of measurement values: fK / fVH (%)

1.0 2.5 6.8 14.9 27.5 42.2

Percentages with respect to fK / fVH (pH 8.0) (%)

2.4 5.9 16.1 35.3 65.2 100.0

As is evident from Table 1, it was found that both the fK / fV values and the fK / fVH values in Example 1 were considerably lower than the corresponding values in Comparative Example 1, which revealed that the

The present process provided a specificity enhanced by fV and fVH and a reduced reactivity by fructosyl-lysines.

[Example 2] Fructosyl-amino acid measurement

35 (1) Sample preparation

Similarly to Example 1, fructosyl-valylhistidine (fVH) (product of BioQuest) and εfructosyl-lysine (fK) (product of Kikkoman Corporation) were dissolved individually in 0.02 mol / l phosphate buffer (pH 7.0) to prepare samples (0.3 mmol / l each).

40

(2) Sample measurement

The prepared samples were measured by means of a HITACHI 7150 automatic analyzer according to the following procedure.

45 <First reagent>

The first reagent was used in Example 1.

50 <Second reagent>

The second reagent used in Example 2 had the same composition as that used in Example 1, except that the 5 U / ml of fructosyl peptide oxidase (FPOX-CE) was exchanged for 5 U / ml of fructosyl peptide. oxidase (FPOX-EE).

55 The first reagent and the second reagent used the same pH for the assay.

The procedure of Example 1 was repeated. The results are shown in Table 2. [Table 2]

pH of the reaction mixture

Example 3 Comparative Example 3

5.5

6.5 8.0

(fV + fK) / fV (%)

101.6 106.2 138.0

(fVH + fK) / fVH (%)

101.7 108.8 141.6

5 As is evident from Table 2, when fructosyl peptide oxidase (FPOX-EE) was used instead of fructosylpeptide oxidase (FPOX-CE) used in Example 1 it was found that the fK / fVH values were considerably lower to the corresponding values in Comparative Example 3, revealing that the present process provides a specificity enhanced by fructosyl-valylhistidine and a reduced reactivity by fructosyl-lysines.

10 [Example 3] Measurement of fructosyl amino acid and fructosyl peptide

(1) Sample preparation

15 Fructosyl-valine, fructosyl-valylhistidine (these two are products of BioQuest) and εfructosyl-lysine (product of Kikkoman Corporation) were dissolved individually in 0.02 mol / l phosphate buffer (pH 7.0) to prepare solutions ( 0.6 mmol / l each). To an aliquot of each of the solutions of 0.6 mmol / l of fructosyl-valine and an aliquot of each of the solutions of 0.6 mmol / l of fructosyl-valylhistidine was added an equivalent amount of 0.02 mol / l phosphate buffer (pH 7.0) to prepare samples 0.3 mmol / l fructosyl valine and

20 samples 0.3 mmol / l of fructosyl-valylhistidine. Separately, an equivalent amount of 0.6 mmol / l of solution was added to an aliquot of each of solutions of 0.6 mmol / l of fructosyl-valine and an aliquot of solutions of 0.6 mmol / l of fructosyl-valinehistidine ε-fructosyl-lysine that had the same amount in order to prepare samples containing fructosyl-valine (0.3 mmol / l) and ε-fructosyl-lysine (0.3 mmol / l), and samples containing fructosyl-valylhistidine ( 0.3 mmol / l) and ε-fructosyl-lysine (0.3 mmol / l).

25

(2) Sample measurement

The prepared samples were measured by means of a HITACHI 7150 automatic analyzer according to the following procedure.

30 <First reagent>

Reagents (pH: 5.5, 6.5, and 8.0) were selected from among the first reagents used in Example 1 and Comparative Example 1 and were used.

35 <Second reagent>

The reagents (pH: 5.5, 6.5, and 8.0) were selected from the second reagents used in Example 1 and Comparative Example 1 and were used.

In each sample, the pH of the first reagent was the same as that of the second reagent.

In a manner similar to that used in Example 1, the values of (fV + fK) / fV (ratio of measurement values of samples containing fructosyl-valine and ε-fructosyl-lysine with respect to the measurement values of

45 samples of fructosyl-valine) and the values of (fVH + fK) / fVH (ratio of measured values of samples containing fructosyl-valylhistidine and ε-fructosyl-lysine with respect to the measured values of fructosylvalylhistidine samples) and evaluated. The results are shown in Table 3.

[Table 3]

pH of the reaction mixture

Example 3 Comparative Example 3

5.5

6.5 8.0

(fV + fK) / fV (%)

101.6 106.2 138.0

(fVH + fK) / fVH (%)

101.7 108.8 141.6

As is evident from Table 3, it was found that the effect of ε-fructosyl-lysine in Example 3 was considerably lower than that of ε-fructosyl-lysine in Comparative Example 3.

[Example 4] Measurement of% hemoglobin A1c (HbA1c)

(1) Sample preparation

5 Samples of whole blood were taken by a routine procedure of 15 subjects using a blood collection tube containing an anticoagulant EDTA. The whole blood samples thus obtained were allowed to stand in a cold room overnight, so that the erythrocytes were allowed to precipitate. An aliquot (10 μl) of each of the erythrocytes so precipitated was sampled. Subsequently 0.1% aqueous Triton X-100 solution (300 μl) was added and mixed with the erythrocytes so sampled (10 μl) to prepare a

10 hemolysis sample.

(2) Sample measurement

The prepared samples were measured by means of a HITACHI 7150 automatic analyzer according to the following procedure.

<First reagent>

1 U / ml proteinase K

20 2 mmol / l WST-3 (product of Dojindo Laboratories) 0.02 mol / l phosphate buffer (pH 8.0)

<Second reagent>

25 4 U / ml fructosyl peptide oxidase (FPOX-CE, product of Kikkoman Corporation) 20 U / ml of peroxidase (III) (product of Toyobo, Ltd.) 80 μmol / l of DA-64 (product of Wako Pure Chemical Industries, Ltd.)

7,500 U / ml of Toyozyme NEP (product of Toyobo, Ltd.) * 37.5 mmol / l of 0.2 mol / l NaCl phosphate buffer (Example 4: pH 6.0, Comparative example 4: pH 8, 0)

30 * Before use, Toyozyme NEP (100,000 U / ml) was dialyzed against 20 mmol / l phosphate buffer (Example 4: pH 6.0, Comparative Example 4: pH 8.0) containing 500 mmol / l of NaCl at 4 ° C for 4 hours. ** The final pH of the reaction mixture of the present invention was 6.7, and that of the reaction mixture of Comparative Example was 8.0.

Each 240 μl of the first reagent was added to each 20 μl of the samples, and the resulting mixture was incubated at 37 ° C for 5 minutes. Subsequently the absorbance of the mixture was measured (absorbance III). The second reagent (80 µl) was added thereto, and the resulting mixture was incubated at 37 ° C for 5 minutes. Subsequently, the absorbance of the mixture was measured (absorbance IV). For each sample, the difference between absorbance III and absorbance IV (difference = absorbance V), which was attributable to a quantity of fructosyl peptide, was calculated using the

40 following formula B.

Equation B: Absorbance V = Absorbance IV - (Absorbance III x (20 + 240) / (20 + 240 + 80))

The absorbance III above is proportional to the total amount of hemoglobin contained in a sample. So,

The absorbance III and absorbance V of the sample were compared with those obtained by the above procedure from a hemolyzed solution of hemocytes having a known hemoglobin A1c (%) value (hemoglobin A1c: 8.6%) in order to Calculate the hemoglobin A1c value (%) of the sample. The hemoglobin A1c values obtained in Example 4 and Comparative Example 4 were compared with that obtained by using a commercially available kit "Rapidia A1c" (product of Fujirebio Inc., based on the procedure of

50 latex) (reference procedure). The results are shown in Table 4.

[Table 4] 5

Sample No.

HbA1c value (%)

Example 4

Comparative Example 4 Reference Example

one

5.2 10.0 6.0

2

4,5 -17.0 5.8

3

5.4 -7.2 6.9

4

5.0 -4.4 5.7

5

4,5 -31.4 5.2

6

3.9 -22.4 4.7

7

4.4 -28.7 4.8

Sample No.

HbA1c value (%)

Example 4

Comparative Example 4 Reference Example

8

5.5 -8.3 7.0

9

5.9 5.6 7.2

10

6.8 5.9 7.5

eleven

6.8 -2.9 8.4

12

9.1 49.1 8.8

13

8.2 23.9 8.7

14

8.9 27.1 9.6

fifteen

11.5 11.5 11.5

Correlation coefficient

0.97 0.74

10

fifteen

twenty

25

30

35

40

Four. Five

As is evident from Table 4, with the procedure of Comparative Example 4, unrealistic negative HbAlc values (%) resulted, while HbAlc values (%) obtained by the method according to the present invention showed good correlation with those of the procedure. reference, as compared to Comparative Example 4. These results show that the process according to the present invention allows the determination of the hemoglobin A1c (%) value in a sample, while preventing the effect of ε-fructosyl-lysine

or fructosyl-lysyl-peptide derived from hemoglobin.

[Example 5] Measurement of the hemoglobin A1c value (%)

(one)

 Preparation of sample

Hemolytic hemolytic samples were prepared from 35 human hemocyte samples in a manner similar to that used in Example 4.

(2)

 Sample Measurement

The prepared samples were measured by means of a HITACHI 7150 automatic analyzer according to the following procedure.

<First reagent>

1 U / ml proteinase K 0.2% of Plysurf A208B (product of DAI-ICHI KOGYO SEIYAKU Co., Ltd.) 0.02 mol / l phosphate buffer (pH 8.0)

<Reactive seconds>

4 U / ml fructosyl peptide oxidase (FPOX-CE, product of Kikkoman Corporation) 20 U / ml peroxidase (III) (product of Toyobo, Ltd.) 80 μmol / l of TPM-PS (product of Dojindo Laboratories)

7,500 U / ml of Toyozyme NEP (product of Toyobo, Ltd.) * 37.5 mmol / l NaCl 0.2 mol / l phosphate buffer (pH: 5.5, 6.0, 7.0 and 8.0) * Before use, each Toyozyme NEP (100,000 U / ml) was dialyzed against 20 mmol / l phosphate buffer (pH: 5.5, 6.0, 7.0 and 8.0 respectively) containing 500 mmol / l NaCl at 4 ° C for 4 hours.

Each 240 μl of the first reagent was added to each 20 μl of the samples, and the absorbance of the resulting mixtures was measured at 600 nm (absorbance VI) by comparison with the reagent blank, which is treated in the same way except that used physiological saline instead of sample. Subsequently, these samples and the control were incubated at 37 ° C for 5 minutes, and the second reagent (80 µl) was added thereto, followed by incubation at 37 ° C for 5 minutes. The absorbance of each of the resulting mixtures was measured at 600 nm using the reagent blank as a control (absorbance VII). It was found that these four reaction mixtures had a final pH of 6.4, 6.7, 7.2 and 8.0, respectively. Hemoglobin A1c (%) values were calculated in a manner similar to that used in Example 4. Separately, as a reference example, hemoglobin A1c (%) values of each of the reaction mixtures were measured using the commercially available kit "RAPIDIA A1c" (product of FUJIREBIO inc.) according to the manufacturer's instructions. Fig. 1 shows the correlation between the hemoglobin A1c (%) values obtained from Example 5 and those obtained from the reference procedure, and the correlation between the hemoglobin A1c (%) values obtained from Comparative Example 5 and those obtained from the procedure reference. Table 5 shows the correlation coefficients between the hemoglobin A1c (%) values obtained from this procedure and those obtained from the reference procedure, and the correlation coefficients between the hemoglobin A1c (%) values obtained from Comparative Example 5 and the obtained from the reference procedure, in addition to average values of percentages (% bias) of the difference between a measurement of Example 5 or Comparative Example 5 and a corresponding measurement of the reference procedure, with respect to the measurement of the reference procedure.

[Table 5]

Example 5

Comparative Example 5

final pH

6.4 6.7 7.2 8.0

Correlation coefficient

0.97 0.96 0.93 0.93

% average bias

-6.4 -8.6 -23.4 -17.9

10 The present measurement procedure proved to be very in accordance with the reference procedure. When the present method is used, hemoglobin A1c (%) in a sample can be measured without being affected by fructosyl-lysylpeptide or ε-fructosyl-lysines derived from hemoglobin.

[Example 6] Identification of the stability during storage of the enzyme for the measurement of fructosyl15 peptide or fructosyl-amino acid

(1) Preparation of the enzyme solution

Enzyme solutions were prepared by the following provisions.

20 50 mmol / l buffer (pH 6.0) * * Buffer agents used: three buffers of potassium phosphate, sodium citrate and N- (2acetamide) iminodiacetic acid (ADA) 5 U / ml of peroxidase (III) ( product of Toyobo, Ltd.)

25 1 U / ml fructosyl peptide oxidase (FPOX-CE, product of Kikkoman Corporation)

(2) Enzyme solution storage

The three types of enzyme solutions prepared above were allowed to stand at 4 ° C or 37 ° C overnight.

(3) Measurement of enzyme activity of enzyme solutions

The enzymatic activity of the enzyme solutions was measured by means of a HITACHI 35 7170 automatic analyzer according to the following procedure.

<Diluted enzyme solution>

Three types of enzyme solutions that had been diluted twice with 100 mmol / l of 40 phosphate buffer (pH 7.5) were used in the measurement.

<First reagent for the measurement of enzymatic activity>

0.5 mmol / l of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine 45 (TOOS)

0.5 mmol / l 4-aminoantipyrine 1 U / ml peroxidase (III) (product of Toyobo, Ltd.) 50 100 mmol / l phosphate buffer (pH 7.5)

<Second reagent for the measurement of enzymatic activity>

150 mmol / l aqueous fructosyl glycine solution

Each 182 μl of the first reagent for the measurement of enzyme activity was added to each 6.5 μl of the diluted enzyme solutions and the resulting mixture was incubated at 37 ° C for 5 minutes. Subsequently, every 26 μl of the second reagents for the measurement of enzymatic activity were added to the reaction mixture. Two to three minutes after the addition of the second reagent, the change in absorbance at 546 nm was measured. For the

The activity evaluation was used, as expressed in%, of the change in absorbance obtained from the enzyme solution that had been incubated at 37 ° C to the change in absorbance obtained from the enzyme solution that had been incubated at 4 ° C (100%)

The relative activity values obtained through the use of potassium phosphate buffer, sodium citrate buffer and ADA buffer were found to be 95%, 98% and 89%, respectively, revealing that these buffers are excellent in stability during storage of the fructosyl peptide or fructosyl amino acid test enzyme.

Claims (13)

one.

A method for reducing the effect of a fructosyl-lysine compound in the fructosyl-peptide or fructosyl-amino acid test, characterized in that it comprises having a fructosyl-peptide-oxidase act on fructosyl-peptide or fructosyl-amino acid at a pH of 4 , 0 to 7.0 and measure the resulting product at a pH of 4.0 to 7.0.

2.

A method for reducing the effect of a fructosyl-lysine compound in the test of a glycosylated protein contained in a sample containing the glycosylated protein, characterized in that it comprises treating the sample with a protease in order to release fructosyl-peptide or free fructosyl-amino acid , make a fructosylpeptide oxidase act on the release of fructosyl peptide or fructosyl amino acid at a pH of 4.0 to 7.0 and measure the resulting product at a pH of 4.0 to 7.0.

3.

The method according to claim 2, wherein the glycosylated protein is glycosylated hemoglobin.

Four.

The method according to claim 2 or 3, wherein the protease is derived from a microorganism belonging to the genus Bacillus, Aspergillus or Streptomyces or is obtained from a gene of the microorganism by a gene recombination technology.

5.

The process according to any one of claims 1 to 4, wherein the fructosyl peptide is fructosylvalylhistidine.

6.

The process according to any one of claims 1 to 4, wherein the fructosyl amino acid is fructosylvaline.

7.

The process according to any one of claims 1 to 6, wherein the product is hydrogen peroxide.

8.

A method for reducing the effect of a fructosyl-lysine compound in the fructosyl-peptide or fructosyl-amino acid test, characterized in that it comprises having at least the following (A) to (C) act on fructosyl-peptide or fructosyl-amino acid at a pH of 4.0 to 7.0

(TO)

a fructosyl peptide oxidase,

(B)

 a reagent for measuring hydrogen peroxide, and

(C)

 an oxidant and glucosone breakdown enzyme.

9.

A method for reducing the effect of a fructosyl-lysine compound in the glycosylated protein assay contained in a sample, characterized in that it comprises treating the sample with a protease to thereby release fructosyl-peptide or fructosyl-amino acid and cause at least the (A) to (C) following on the release of fructosyl-peptide or fructosyl-amino acid at a pH of 4.0 to 7.0:

(TO)

a fructosyl peptide oxidase,

(B)

 a reagent for measuring hydrogen peroxide, and

(C)

 an oxidant and glucosone breakdown enzyme.

10.

The method according to claim 9, wherein the glycosylated protein is glycosylated hemoglobin.

eleven.

The method according to claim 9 or 10, wherein the protease is derived from a microorganism belonging to the genus Bacillus, Aspergillus or Streptomyces or is obtained from a gene of the microorganism by a gene recombination technology.

12.

The process according to any one of claims 8 to 11, wherein the fructosyl peptide is fructosylvalylhistidine.

13.

The process according to any one of claims 8 to 11, wherein the fructosyl amino acid is fructosyl valine.